This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Shotgun proteomics techniques typically "undersample" peptides in complex mixtures using data-dependent acquisition (DDA). Multi-dimensional chromatographic separations have been used to decrease the number of co-eluting ions, allowing the DDA process to sample the mixtures adequately. However, the mass spectrometer stills spend a majority of experiment time sampling the most abundant species. In data-independent acquisition (DIA), windows in the m/z domain are fragmented continuously, providing an opportunity to obtain fragmentation spectra at every m/z value of interest, without any m/z bias. This process can be improved by increasing the ion flux, which improves sampling of less abundant molecular species. This project investigates improvements in complex mixture analysis by using DIA on a mass spectrometer equipped with an electrodynamic ion funnel. The ion funnel, built in-house from a design by researchers at Pacific Northwest National Lab, increases ion flux by a factor of 10 or more, leading to increased scan speed, sensitivity, and increased numbers of peptide and protein identifications, in multiple modes of analysis.